1. Technical Field of the Invention
The present invention relates to a method for measuring the protease activity of transglutaminase-containing products that are useful as enzyme additives in protein-containing materials, and to methods for preparing transglutaminase formulation for binding application. In particular, the present invention relates to a method for measuring the protease activity in transglutaminase-containing products, this protease activity being closely related to the functioning of transglutaminase in binding application and Surimi products; methods of preparing transglutaminase-containing products in which the protease activity is regulated based on the value of the protease activity measured by this measurement method; and transglutaminase formulations, manufactured according to these preparation methods for binding application and Surimi products, in which protease activity is regulated.
2. Background Description
Transglutaminase is an enzymatic substance known to modify the physical properties of protein-containing materials. It is added directly to protein-containing materials to modify their physical properties, or is mixed with protein-containing materials, particularly gelatins and milk protein solutions, and employed in various substances as binding composition. However, transglutaminase formulations manufactured using available transglutaminase-containing products, particularly transglutaminase-containing products derived from microbes, vary greatly in quality from lot to lot in the same manner as other enzymatic formulations.
The principal reason for the variation between lots in the effectiveness of transglutaminase formulations in protein-containing materials is thought to be variation in the transglutaminase activity of the transglutaminase-containing product. However, variation in quality remains even when transglutaminase activity is controlled, constituting a major operational problem.
There have also been cases in which the presence of protease has been suspected of causing change in protein-containing materials to which transglutaminase-containing products have been added. However, the correlation between change and protease activity in transglutaminase-containing products measured by conventional methods of measuring protease activity has been extremely low, and there have not been any cases in which protease has been determined to be the cause. Since the cause of the variation in quality has been, unclear in this manner, ensuring the quality of a formulation has required countermeasures such as sorting by testing and increasing the blending ratio of transglutaminase-containing product to achieve a surplus. However, both of these methods entail increased labor and starting material costs, driving up the cost of manufacturing a formulation. Thus, once the reason is discovered, there is great need for a quality control technique based on an index with a high correlation with quality for both transglutaminase-containing products and transglutaminase formulations manufactured using such products.
Examples of undesired protease being present in or contaminating a protein-containing material, thereby negatively affecting the material, have been known for some time. However, the quantity producing an effect varies with the type and use of protein-containing materials in which this occurs, as well as with the type and origin of the protease. Further, a number of methods of measuring protease activity have been proposed. However, the correlation between the protease activity value that is measured and the magnitude of the effect on the protein-containing material is not necessarily high. Thus, these methods have afforded low reliability as quality control methods for protease activity.
In transglutaminase derived from microbes, there is a known example of the intentional formulation of protease in prescribed applications (Japanese Patent Application Publication No. H07-023740). An example of an attempt to control the effect of residual protease in a protein-containing material enhanced with transglutaminase is also known (Japanese Patent Application Publication No. H9-299065). However, there is no known case of measuring the effect on food of protease present as an impurity in a transglutaminase-containing product and attempting to regulate it.
Known methods of selectively deactivating protease in enzyme-containing products include a method of eliminating the activity of enzymes present as impurities, such as protease and α-amylase, by a 16 hour treatment at 37° C. and pH 9.8 (Japanese Patent Application Publication No. H11-42086) and a method of deactivating the protease present in lactase by irradiation with γ-rays (Japanese Patent Application Publication No. S54-18349).
However, there is no description suggesting any relation with effects such as the binding strength of transglutaminase-containing products in which protease activity has been decreased by these methods or improved physical properties in Surimi products.
There is literature (2nd Ed., Self-Imposed Standards for Food Additives Other than Chemically Synthesized Products, published by the Japan Food Additive Association, pp. 215-223 (1993)) relating to methods of measuring the activity of common proteases (papain, pancreatin, bromelain, and pepsin). However, there is no description suggesting any correlation between the value of the protease activity in a transglutaminase-containing product measured by this method and binding strength when this transglutaminase-containing product is employed as a binding component, in a formulation for Surimi products, or to produce a physical property-enhancing effect in Surimi products.